Immunohistochemical Evaluation of Gli1 and HER2

Fix the surgical specimens with formalin and embed them with paraffin. Then the tissues were cut into 5 μm. After dewaxing and rehydrating, sections were incubated with the polyclonal antibodies of Gli1 or HER2 (Cell Signaling Technology, USA; 1:200 dilution) at the room temperature for 2 or 3 hours. The process was performed with the tissue staining kit (Zhongshan Biotechnology, Beijing, China) following the manufacturer’s protocol. Two researchers got the outcome of immunostaining respectively. Five 200 × random regions were analyzed and classified into five levels according to the percentage of positively staining cells per section: absent, 0~5%; 1, 6~25%; 2, 26~50%; 3, 51~75%; 4, >75%. We deemed the staining intensity as follows: 0 (negative); 1 (weak); 2 (moderate); 3 (strong). The percentage and intensity scores were then multiplied to obtain a total score (staining score = percentage score × intensity score). For the staining score, 0 was deemed as (−), 1~4 as (+), 5~8 as (++), 9~12 as (+++). Eventually, we obtained a final average score, and consider (−) or (+) as negative, (++) or (+++) as positive^{41 (link)}.